Systems, Methods, and Apparatus for Imaging of Diffuse Media Featuring Cross-Modality Weighting of Fluorescent and Bioluminescent Sources

ABSTRACT

In certain embodiments, the invention relates to systems and methods for in vivo tomographic imaging of fluorescent probes and/or bioluminescent reporters, wherein a fluorescent probe and a bioluminescent reporter are spatially co-localized (e.g., located at distances equivalent to or smaller than the scattering mean free path of light) in a diffusive medium (e.g., biological tissue). Measurements obtained from bioluminescent and fluorescent modalities are combined per methods described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.14/054,651, filed Oct. 15, 2013, and having the same title, which inturn claims priority to and the benefit of, U.S. Provisional PatentApplication No. 61/714,198, filed Oct. 15, 2012, each of which is herebyincorporated by reference in their entirety.

FIELD

The invention relates generally to in vivo imaging systems and methods.More particularly, in certain embodiments, the invention relates to atomographic imaging system employing co-located fluorescent andbioluminescent sources.

BACKGROUND

Imaging in diffusive media has become an attractive field of researchmainly due to its applications in biology and medicine, showing greatpotential in, for example, cancer research, drug development,inflammation and molecular biology. With the development of highlyspecific activatable fluorescent probes, a new way of obtaininginformation at the molecular level in vivo has been made possible. Dueto the inherent scattering present in tissues in the opticalwavelengths, light is multiply scattered in tissue and its originaldirection of propagation is randomized after what is termed thescattering mean free path, lsc, a distance which represents the densityand efficiency of scattering of the different constituents of tissue.Even though images of fluorescent or bioluminescent emission fromtissues might be recorded, due to the scattering present, there is anon-linear relation of these images with the concentration, size andposition of the fluorescent probes or bioluminescent reporters,resulting in what is termed an ill-posed problem. Recently developedmethods and systems make use of tomographic approaches by introducing aspatial dependence of fluorescence on the excitation and appropriatemodeling of light propagation in tissues through the diffusionapproximation to alleviate this ill-posedness, enabling the recovery ofthe spatial distribution of the concentration of fluorescence with aresolution on the order of the scattering mean free path (lsc) orbetter. In an ideal situation, the sensitivity of these tomographictechniques would depend only on the detector efficiency and probebrightness together with the absorption present in tissue, assuming itis possible to completely block the excitation light when measuringfluorescence. In this ideal case, the sensitivity could be increased byaugmenting the laser power since the emitted and excitation intensitiesare proportional to one another, at least for powers acceptable forsmall animal imaging, which ensure that no appreciable heating occurs intissue.

However, the reality of an in vivo measurement is quite different. Dueto the excitation of surrounding auto-fluorescence always present intissue, the sensitivity of a system is strongly dependent on the abilityto distinguish the specific signal due to the fluorescent probe from thenon-specific signal of the surrounding auto-fluorescence. This problembecomes more pronounced when exciting in reflection mode, since thegreater auto-fluorescent contribution would be from the tissue sectionsclosest to the camera. In practice, the sensitivity of the tomographicdata, or in general any collection of fluorescent data, is determined bythe level of auto-fluorescence when compared to the specific signal.This issue is overcome to some extent by using far-red or near infra-red(NIR) fluorescence signals, since tissue auto-fluorescence is slightlyreduced in this part of the spectrum, with the added advantage thattissue presents lower absorption properties in this part of thespectrum. This is the case when using fluorescent probes that emit inthe far-red or near infra-red part of the spectrum, which are activatedwhen a specific molecular activity is present. An advantage of this kindof probes is that they provide a high signal-to-noise ratio. However, inmost practical instances, the amount of signal that can be detecteddepends on how much specific signal surpasses the surrounding tissueauto-fluorescence.

In order to separate the contribution of the specific from thenon-specific signal, the most common approach is to employmulti-spectral measurements, assuming the emission spectrum is known.Even though this approach slightly increases the sensitivity, theproblem still remains: at each emission wavelength measured there is anunknown contribution from tissue auto-fluorescence. The weaker thespecific signal, the more dominant the effect of auto-fluorescence.

Bioluminescent reporters offer the significant advantage of notrequiring an external illumination to place them in an excited statesuch that they would emit light. Since the excited state is reachedthough a chemical reaction, the emitted light represents thebackground-free solution of the imaging problem, akin to the ideal caseof fluorescence mentioned in the previous paragraphs. This inherentbenefit of bioluminescence also has some drawbacks, specifically inrelation to the ill-posed problem mentioned above. Since it is notcurrently possible to effectively introduce a spatial dependence on theintensity of this emission (whereas this is possible in the case offluorescence), it is not possible to recover simultaneously the spatialdistribution of bioluminescent probe concentration. Thus, because themain goal is to recover the spatial distribution of probe concentration,the ill-posedness of the problem cannot be substantially reduced in thecase of bioluminescence as is possible in the case of fluorescence.

There is a need for an optical imaging system in which thelow-background and lack of auto-fluorescence of bioluminescent probescan be combined with the specificity, high quantum yield, and theexternal capability of emission intensity modulation exhibited byfluorescent probes.

SUMMARY

In certain embodiments, the invention relates to systems and methods forin vivo tomographic imaging of fluorescent probes and/or bioluminescentreporters, wherein a fluorescent probe and a bioluminescent reporter arespatially co-localized (e.g., located at distances equivalent to orsmaller than the scattering mean free path of light) in a diffusivemedium (e.g., biological tissue). Measurements obtained frombioluminescent and fluorescent modalities are combined per methodsdescribed herein.

In one aspect, the invention provides a method for imaging a targetregion of a diffuse object, the method comprising: (a) administering abioluminescent substrate (and/or chemiluminescent substrate) to theobject; (b) detecting bioluminescent (and/or chemiluminescent) lightemitted from the object by a bioluminescent reporter in the targetregion of the object (e.g., using an external detector); (c)administering a probe comprising a fluorescent species (and/or a jointlyfluorescent/bioluminescent species) to the object; (d) directingexcitation light into the object at multiple locations and/or atmultiple angles, thereby exciting the fluorescent species; (e) detectingfluorescent light (e.g., as a function of detector position, excitationlight source position, or both), the detected fluorescent light havingbeen emitted by the fluorescent species in the target region of thediffuse object as a result of excitation by the excitation light; (f)detecting excitation light (e.g., as a function of detector position,excitation light source position, or both), the detected excitationlight having been transmitted through the region of the diffuse object(transillumination) or having been reflected from the region of thediffuse object (epi-illumination); and (g) processing data correspondingto the detected bioluminescent light, the detected fluorescent light,and the detected excitation light to provide an image of the targetregion within the diffuse object.

In certain embodiments, the bioluminescent reporter is endogenous. Incertain embodiments, the bioluminescent reporter is expressed within theobject by a bioluminescent cell line. In certain embodiments, thebioluminescent reporter is exogenously administered. In certainembodiments, the bioluminescent reporter is administered to the objectas a component of a tandem bioluminescent-fluorescent probe.

In certain embodiments, following step (c), at least some of thebioluminescent reporter is substantially co-located with at least someof the (fluorescent) probe in the target region of the object. Incertain embodiments, following step (c), the bioluminescent reporter isalso present in the object at one or more locations that are notsubstantially co-located with the (fluorescent) probe. In certainembodiments, the probe in step (c) comprises both the fluorescentspecies and the bioluminescent reporter in tandem with the fluorescentspecies (e.g., the bioluminescent reporter and fluorescent probecomprises a single construct). In certain embodiments, it is notrequired that the bioluminescent reporter and the fluorescent species beconjugated or colocalized to within FRET-type distances. Co-located maymean located within a macro-level distance. For example, in certainembodiments, the method may be used even where the bioluminescentreporter and the fluorescent species are separated by a macro-leveldistance, for example, up to about 1 mm or up to about 2 mm, rather thanup to about 5 to 10 nm as in FRET and BRET.

In certain embodiments, the diffuse object comprises living biologicaltissue (in vivo imaging). In certain embodiments, the diffuse object isa mammal. In certain embodiments, the image of the target region is atomographic image. In certain embodiments, the method further comprisesplacing the object within a holder prior to steps (b), (d), (e), and(f). For example, in certain embodiments, the image of the target regionis a tomographic image and wherein a surface of the object is at leastpartially conformed by the holder such that the surface can becharacterized by a continuous function (e.g., in Cartesian, polar, orcylindrical coordinates), thereby facilitating tomographicreconstruction.

In certain embodiments, step (b) comprises detecting bioluminescentlight using a detector located outside the object. In certainembodiments, step (b) comprises detecting bioluminescent light as afunction of detector position. In certain embodiments, step (b)comprises detecting bioluminescent light using a detector in opticalcontact with the object (e.g., a component of the detector is inphysical contact with either the object itself or a transparent surfacein physical contact with the object, and/or optical guides, fiberguides, optical matching fluids, and or lenses are used such thatoptical contact is maintained during detection). In certain embodiments,step (b) comprises detecting bioluminescent light using a detectorpositioned such that there is a nondiffusive medium (e.g., a layer ofair) between the detector and the object. In certain embodiments, step(b) comprises detecting bioluminescent light using an emission filter.In certain embodiments, step (b) comprises detecting bioluminescentlight without using an emission filter.

In certain embodiments, the probe in step (c) is a visible ornear-infrared fluorescent probe (e.g., a NIRF molecular probe, a redagent, etc.).

In certain embodiments, step (d) comprises directing visible and/or nearinfrared light into the object. In certain embodiments, step (d)comprises directing a point source of excitation light into the object.In certain embodiments, step (d) comprises simultaneously directingmultiple sources of excitation light into the object. In certainembodiments, step (d) comprises directing structured excitation lightinto the object. In certain embodiments, step (d) comprises directingthe excitation light into the object at multiple discrete positions(e.g., an array of positions) and/or at multiple discrete angles (e.g.,an array of angles), each instance of directing the excitation lightinto the object occurring at discrete times.

In certain embodiments, the method comprises performing steps (e) and(f) after each instance of directing excitation light into the object ata discrete position and/or at a discrete angle. In certain embodiments,step (d) comprises directing the excitation light into the object atmultiple discrete positions (e.g., an array of positions) and/or atmultiple discrete angles (e.g., an array of angles) simultaneously. Incertain embodiments, step (d) comprises scanning the excitation lightover the object. In certain embodiments, the excitation light detectedin step (f) comprises at least one of continuous wave (CW) light,time-resolved (TR) light, and intensity modulated (IM) light.

In certain embodiments, step (e) comprises detecting fluorescent lightemitted from the target region of the object using a detector locatedoutside the object. In certain embodiments, step (e) comprises detectingfluorescent light on the same side of the object into which excitationlight was directed in step (d) (e.g., epi-illumination). In certainembodiments, step (e) comprises detecting fluorescent light on a side ofthe object opposite the side into which excitation light was directed instep (d) (e.g., transillumination). In certain embodiments, step (e)comprises detecting fluorescent light emitted from the target region ofthe object using a detector in optical contact with the object (e.g., acomponent of the detector is in physical contact with either the objectitself or a transparent surface in physical contact with the object,and/or optical guides, fiber guides, optical matching fluids, and orlenses are used such that optical contact is maintained duringdetection). In certain embodiments, step (e) comprises detectingfluorescent light emitted from the target region of the object using adetector positioned such that there is a nondiffusive medium (e.g., alayer of air) between the detector and the object.

In certain embodiments, step (f) comprises detecting excitation lighttransmitted through the target region of the object or reflected fromthe target region of the object using a detector located outside theobject. In certain embodiments, step (f) comprises detecting excitationlight transmitted through the target region of the object or reflectedfrom the target region of the object using a detector in optical contactwith the object (e.g., a component of the detector is in physicalcontact with either the object itself or a transparent surface inphysical contact with the object, and/or optical guides, fiber guides,optical matching fluids, and or lenses are used such that opticalcontact is maintained during detection). In certain embodiments, step(f) comprises detecting excitation light transmitted through the targetregion of the object or reflected from the target region of the objectusing a detector positioned such that there is a nondiffusive medium(e.g., a layer of air) between the detector and the object.

In certain embodiments, step (e) comprises detecting the fluorescentlight from multiple projections and/or views. In certain embodiments,step (f) comprises detecting the excitation light from multipleprojections and/or views.

In certain embodiments, the target region of the object is athree-dimensional region and step (g) comprises providing a tomographicimage that corresponds to the fluorescent species in thethree-dimensional target region. In certain embodiments, the tomographicimage indicates three-dimensional spatial distribution of the probe(e.g., wherein the probe comprises a fluorescent species and/or ajointly fluorescent/bioluminescent species) within the target region. Incertain embodiments, the tomographic image indicates concentration ofthe probe as a function of position within the target region of theobject. In certain embodiments, the tomographic image indicatesconcentration of the probe as a function of position in threedimensions.

In certain embodiments, the target region of the object is athree-dimensional region and step (g) comprises providing a tomographicimage that corresponds to the bioluminescent reporter in thethree-dimensional target region. In certain embodiments, the tomographicimage indicates three-dimensional spatial distribution of thebioluminescent reporter within the target region. In certainembodiments, the tomographic image indicates concentration of thebioluminescent reporter as a function of position within the targetregion of the object.

In certain embodiments, the bioluminescent reporter and the fluorescentspecies have emission spectra that differ.

In certain embodiments, step (g) comprises weighting data correspondingto the detected fluorescent light with data corresponding to thedetected bioluminescent light and normalizing data corresponding to thedetected fluorescent light with data corresponding to the detectedexcitation light. In certain embodiments, step (g) comprises weightingdata corresponding to the detected fluorescent light with datacorresponding to the detected bioluminescent light and then normalizingthe resulting weighted data with data corresponding to the detectedexcitation light, then inverting an associated weight matrix to obtaina/the tomographic image of the fluorescent species in the target regionof the object. In certain embodiments, step (g) comprises weighting datacorresponding to the detected fluorescent light with data correspondingto the detected bioluminescent light, and weighting an associated weightmatrix with data corresponding to the detected bioluminescent light,then inverting the weight matrix to obtain a/the tomographic image ofthe fluorescent species in the target region of the object. In certainembodiments, step (g) comprises weighting data corresponding to thedetected bioluminescent light with data corresponding to the detectedfluorescent light normalized with data corresponding to the detectedexcitation light, then inverting an associated weight matrix to obtaina/the tomographic image of the bioluminescent reporter in the targetregion of the object.

In certain embodiments, the detecting in step (b) is performed at adifferent time than the detecting in steps (e) and (f) (e.g., becausepharmacokinetics of the probes and the substrate may be different). Incertain embodiments, one would time the administration (e.g., injection)of bioluminescent substrate and fluorescent probe such that the imagingof both modalities can be performed in close succession, in order tocapture a single “time point”. Imaging via the two modes need not occurin exact simultaneity, for example, in an instrument equipped to imagein both modalities. It is possible to image using two single-modalityinstruments that are in proximity such that imaging the object (e.g., aliving animal) in one instrument can be performed, and the animal may beshuttled to the second instrument, e.g., within a few minutes. An animalholder may be used, for example, such as that described in U.S. PatentApplication Publication No. US 2011/0071388, incorporated herein byreference.

In another aspect, the invention provides a method for imaging a targetregion of a diffuse object, the method comprising: (a) administering abioluminescent substrate (and/or chemiluminescent substrate) to theobject; (b) detecting bioluminescent (and/or chemiluminescent) lightemitted from the object by a bioluminescent reporter in the targetregion of the object (e.g., using an external detector); (c)administering a probe comprising a fluorescent species (and/or a jointlyfluorescent/bioluminescent species) to the object; (d) detectingfluorescent light (e.g., as a function of detector position), thedetected fluorescent light having been emitted by the fluorescentspecies in the target region of the diffuse object as a result ofexcitation by the bioluminescent light (e.g., bioluminescent stimulationof fluorescence by radiative transfer); and (e) processing datacorresponding to the detected bioluminescent light and the detectedfluorescent light to provide an image of the target region within thediffuse object.

In certain embodiments, the method further includes directing light(e.g., at an excitation wavelength) into the object and detecting thelight having been transmitted through the region of the diffuse object(transillumination) or having been reflected from the region of thediffuse object (epi-illumination) (e.g., to provide a proxy attenuationmap), and using data corresponding to the detected light (e.g., usingthe proxy attenuation map) in step (g) along with the data correspondingto the detected bioluminescent light and the detected fluorescent lightto provide the image of the target region within the diffuse object.

In another aspect, the invention provides a system for imaging a targetregion within a diffuse object, the system comprising: an excitationlight source; an optical imaging apparatus configured to direct lightfrom the excitation light source into the diffuse object at multiplelocations and/or at multiple angles; one or more detectors, the one ormore detectors individually or collectively configured to detect and/ormeasure (e.g., as a function of detector position, excitation lightsource position, or both), (i) bioluminescent (and/or chemiluminescent)light emitted from the object, (ii) fluorescent light emitted by afluorescent species in the target region of the diffuse object as aresult of excitation by the excitation light, and (iii) excitation lighthaving been transmitted through the region of the diffuse object orhaving been reflected from the region of the diffuse object; and aprocessor configured to process data corresponding to the detectedbioluminescent light, the detected fluorescent light, and the detectedexcitation light to provide an image of the target region within thediffuse object.

In another aspect, the invention provides a system for imaging a targetregion within a diffuse object, the system comprising: an excitationlight source; an optical imaging apparatus configured to direct lightfrom the excitation light source into the diffuse object at multiplelocations and/or at multiple angles; one or more detectors, the one ormore detectors individually or collectively configured to detect and/ormeasure (e.g., as a function of detector position, excitation lightsource position, or both), (i) bioluminescent (and/or chemiluminescent)light emitted from the object, (ii) fluorescent light emitted by afluorescent species in the target region of the diffuse object as aresult of excitation by the excitation light, and (iii) excitation lighthaving been transmitted through the region of the diffuse object orhaving been reflected from the region of the diffuse object; and aprocessor configured to process data corresponding to the detectedbioluminescent light, the detected fluorescent light, and the detectedexcitation light to provide an image of the target region within thediffuse object.

In another aspect, the invention provides an apparatus forreconstructing a tomographic representation of a probe within a targetregion of a diffuse object, the apparatus comprising: a memory thatstores code defining a set of instructions; and a processor thatexecutes the instructions thereby to process data corresponding to: (a)establish a forward model of excitation light propagation from anexcitation light source to the probe within the target region of theobject and of emission light propagation from the probe to a detectorusing (i) data corresponding to detected fluorescent light from theprobe, (ii) data corresponding to detected excitation light having beentransmitted through the region of the diffuse object or having beenreflected from the region of the diffuse object, and (iii) datacorresponding to detected bioluminescent light emitted from abioluminescent reporter, wherein at least some of the bioluminescentreporter is substantially co-located with at least some of the(fluorescent) probe in the target region of the object, and wherein theforward model is established as a weight matrix; and (b) invert theweight matrix to obtain the tomographic representation of the probewithin the target region of the diffuse object.

In another aspect, the invention provides a non-transitory computerreadable medium having instructions thereon that, when executed by aprocessor, cause the processor to: (a) establish a forward model ofexcitation light propagation from an excitation light source to theprobe within the target region of the object and of emission lightpropagation from the probe to a detector using (i) data corresponding todetected fluorescent light from the probe, (ii) data corresponding todetected excitation light having been transmitted through the region ofthe diffuse object or having been reflected from the region of thediffuse object, and (iii) data corresponding to detected bioluminescentlight emitted from a bioluminescent reporter, wherein at least some ofthe bioluminescent reporter is substantially co-located with at leastsome of the (fluorescent) probe in the target region of the object, andwherein the forward model is established as a weight matrix; and (b)invert the weight matrix to obtain the tomographic representation of theprobe within the target region of the diffuse object.

In another aspect, the invention provides a method for imaging a targetregion of a diffuse object, the method comprising: (a) detectingbioluminescent (and/or chemiluminescent) light emitted from the objectby a bioluminescent reporter in the target region of the object (e.g.,using an external detector); (b) directing excitation light into theobject at multiple locations and/or at multiple angles, thereby excitinga fluorescent species of a probe (e.g., a fluorescent probe or afluorescent/bioluminescent tandem probe) in the target region of thediffuse object; (c) detecting fluorescent light as a function ofdetector position, excitation light source position, or both, thedetected fluorescent light having been emitted by the fluorescentspecies as a result of excitation by the excitation light; (d) detectingexcitation light as a function of detector position, excitation lightsource position, or both, the detected excitation light having beentransmitted through the region of the diffuse object or having beenreflected from the region of the diffuse object; and (e) processing datacorresponding to the detected bioluminescent light, the detectedfluorescent light, and the detected excitation light to provide an imageof the target region within the diffuse object.

Elements from embodiments of one aspect of the invention may be used inother aspects of the invention (e.g., elements of claims depending fromone independent claim may be used to further specify embodiments ofother independent claims). Other features and advantages of theinvention will be apparent from the following figures, detaileddescription, and the claims.

The objects and features of the invention can be better understood withreference to the drawings described below, and the claims. In thedrawings, like numerals are used to indicate like parts throughout thevarious views.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic drawing depicting a comparison between an imagegenerated by a bioluminescent source and one generated by a fluorescencesource, the intensity of which can be modulated externally by changingthe intensity of the excitation source. The shape of both signals shouldbe, in the absence of auto-fluorescence, identical, with the onlydifference residing in their absolute intensity values.

FIG. 2 is a schematic drawing depicting how the information due solelyto the fluorophore can be identified with the bioluminescence profilewhen the fluorescence signal is degraded through contribution ofauto-fluorescence and background signal.

FIG. 3 is a schematic drawing depicting how the contribution ofauto-fluorescence changes with the source position, while thefluorescence emission changes in intensity but not in distribution.

FIG. 4 is a block flow diagram depicting an example method of combiningbioluminescence with normalized fluorescent measurements to obtain 3Dimages of fluorescent and/or bioluminescent sources, according to anillustrative embodiment of the invention.

FIG. 5 shows the results of a simulation of the combination ofbioluminescence with normalized fluorescent measurements to obtainaccurate 3D images of fluorescent sources in the presence ofnon-specific background fluorescent signal, according to an illustrativeembodiment of the invention.

FIG. 6 shows a schematic of a diffuse optical tomography imaging systemthat may be used in various embodiments described herein.

DETAILED DESCRIPTION

It is contemplated that methods, systems, and processes described hereinencompass variations and adaptations developed using information fromthe embodiments described herein.

Throughout the description, where systems and compositions are describedas having, including, or comprising specific components, or whereprocesses and methods are described as having, including, or comprisingspecific steps, it is contemplated that, additionally, there are systemsand compositions of the present invention that consist essentially of,or consist of, the recited components, and that there are processes andmethods of the present invention that consist essentially of, or consistof, the recited processing steps.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim.

Headers are used herein to aid the reader and are not meant to limit theinterpretation of the subject matter described.

As used herein, the term “image” is understood to mean a visual displayor any data representation that may be interpreted for visual display.For example, a three-dimensional image may include a dataset of valuesof a given quantity that varies in three spatial dimensions. Athree-dimensional image (e.g., a three-dimensional data representation)may be displayed in two-dimensions (e.g., on a two-dimensional screen,or on a two-dimensional printout).

The term “tomographic image” may refer, for example, to an opticaltomographic image, an x-ray tomographic image, a tomographic imagegenerated by magnetic resonance, positron emission tomography (PET),magnetic resonance, (MR) single photon emission computed tomography(SPECT), and/or ultrasound, and any combination of these.

As used herein, the term “detector” includes any detector ofelectromagnetic radiation including, but not limited to, CCD camera,photomultiplier tubes, photodiodes, and avalanche photodiodes.

As used herein, the term “forward model” is understood to mean aphysical model of light propagation in a given medium, for example, froma source to a detector.

Described herein are methods of measurement and analysis that combineinformation obtained from co-existent bioluminescence and fluorescentsignals in diffuse media. These methods increase the accuracy oftomographic reconstructions in diffuse media and enhance thequantitative, three-dimensional structural, functional and molecularinformation obtained in in vivo imaging studies in small animals. Thisincrease in accuracy is obtained by reducing the ill-posedness ofbioluminescent imaging in order to obtain three-dimensional (3D)reconstructions from measurements of bioluminescence signals with theaid of fluorescence.

The bioluminescent signal and the probe-specific fluorescent signal inthe absence of a background signal should be equivalent in morphologybut not in intensity. It is the intensity of the fluorescence emissionthat provides the 3D information needed for the reconstruction, whilethe bioluminescence emission provides the background-free spatial signaldistribution. By combining detection of both bioluminescence andfluorescence, it is possible to retrieve 3D information of fluorescentprobes with the superior signal-to-noise ratio of bioluminescence due tothe lack of background signal. In some embodiments, this inventionfurther enables the use of analytical tools to combine the informationobtained by bioluminescence and fluorescence measurements, resulting inimproved feasibility of the imaging reconstructions from both types oflight sources. These tools will provide 3D information to in vivo animaloptical imaging setups through the use of normalized fluorescencemeasurements, while simultaneously providing auto-fluorescence-freemeasurements with the aid of bioluminescence.

One of the main analytical aspects when imaging diffuse media is thefeasibility of simultaneously recovering information on the position,concentration, and distribution (or size) of a collection oflight-emitting reporters (i.e., fluorescent or bioluminescent). In thecase of a single excitation source or in its absence, as in the case ofbioluminescence, there is an infinite combination of concentration,position, and size that will yield exactly the same measurement, whichresults in an ill-posed problem. The nature of this ill-posedness isfundamentally represented by the way information is lost during lightpropagation, as indicated by the transfer function, a function whichrelates the spatial distribution of intensity in Fourier space for thespatial frequencies K at a plane z′ with the spatial distribution ofintensity at another plane z:

H(K,z−z′)=exp(iq(K)|z−z′|)  (1)

One way of reducing the ill-posedness of the problem is to change theintensity of the emission in a controlled manner, so that a spatialdependence is introduced. In the case of fluorescence, this can be donedirectly through the excitation source, Uexc(r):

S(r)=σ_(abs)[C(r)ϕ(λ_(em))U _(exc)  (2)

where σ_(abs) represents the absorption cross-section of thefluorophore, [C(r)] its concentration, Φ(λ_(em)) its quantum yield, andU_(exc)(r) the spatial distribution of excitation intensity. However,this may not be done for bioluminescence. Since a spatial dependencecannot be introduced on the emission of a bioluminescent reaction, theproblem of recovering the position, concentration, or size of abioluminescent probe will remain extremely ill-posed. This is a majordrawback of bioluminescence.

In certain embodiments, methods described herein use the informationobtained from coexistent bioluminescent and fluorescent tomographicsignals co-localized at distances similar to or shorter than thescattering mean free path lsc. This leads to two important consequences.First, it allows the use of the spatial dependence introduced by thefluorescent excitation source to reduce the ill-posedness of thebioluminescent imaging. This has a direct impact in the recovery ofbioluminescent probe spatial position and size, allowing a 3Dreconstruction of the bioluminescent data obtained with the aid offluorescent measurements. Second, this method allows the use of the highspecificity of the bioluminescent signal to improve the accuracy of thetomographic reconstructions of the co-localized fluorescent signal, byproviding information that will allow the discrimination of specificfluorescent signal from background or auto-fluorescence signal. Thisincreases and improves the resolution, quantification, and mostimportantly, the sensitivity of fluorescence tomography.

The methods can be used, for example, where a tandembioluminescence-fluorescence probe, or single or multiple co-localizedbioluminescence reporter(s) and fluorescence probe(s) are used in thepresence or absence of background auto-fluorescence or non-specificsignal (such as excitation light leaking through the emission filters).Three examples of the approaches that can be used to combine thebioluminescence and fluorescence measurements are presented below,showcasing the feasibility of the method, but not limiting it to theseapproaches. A fourth example below describes an approach for acquiringthe data that are used in the other examples, but does not limit theapplication to this approach.

Bioluminescence Weighted Fluorescence Data

As a first approach, a weight is introduced on fluorescence measurementsby assuming that for each source position r_(s) and detector r_(d)represented by U^(fl)(r_(s),r_(d)), there is a measured distribution ofbioluminescence U^(bio)(r_(d)) (there is only a dependence on thedetector in the case of bioluminescence). In this case, an exampleapproach would be to determine the weight as:

$\begin{matrix}{{h\left( r_{d} \right)} = \frac{U^{bio}\left( r_{d} \right)}{{mean}\mspace{14mu} \left\{ {U^{bio}\left( r_{d} \right)} \right\}}} & (3)\end{matrix}$

which would ensure that the weights are within the [0,1] range. Thefluorescence measurements to be used for the reconstruction would be asfollows:

U _(w) ^(fl)(r _(s) ,r _(d))=U ^(fl)(r _(s) ,r _(d))h(r _(d))  (4)

There are a variety of ways of choosing the weight h. Another approachis to set a threshold in the measurement, so that h is defined as:

$\begin{matrix}{{h\left( {r_{d},{thresh}} \right)} = \frac{{U^{bio}\left( r_{d} \right)}{H\left( {{U^{bio}\left( r_{d} \right)} > {thresh}} \right)}}{{mean}\mspace{14mu} \left\{ {U^{bio}\left( r_{d} \right)} \right\}}} & (5)\end{matrix}$

where H is the Heavyside function, which would yield 1 for values of themeasurement above the threshold. This threshold will propagate to allfluorescence measurements, in which case the actual values used for thefluorescence reconstruction do not depend on the fluorescence signalmeasured but rather on the case of having actual bioluminescent (andtherefore noise-free) signal or not. Once the weighted fluorescencemeasurement is defined, the regular reconstruction method is thenperformed, first normalizing the weighted fluorescence by theexcitation, U^(exc)(r_(s),r_(d)):

$\begin{matrix}{{U_{nw}\left( {r_{s},r_{d}} \right)} = {\frac{U_{w}^{fl}\left( {r_{s},r_{d}} \right)}{U^{exc}\left( {r_{s},r_{d}} \right)} = {\frac{U^{fl}\left( {r_{s},r_{d}} \right)}{U^{exc}\left( {r_{s},r_{d}} \right)}{h\left( r_{d} \right)}}}} & (6)\end{matrix}$

This weighted normalized fluorescence can be used to recover thenoise-free (or specific) fluorophore distribution, F(r) by inverting theequation:

U _(nw)(r _(s) ,r _(d))=∫_(V) W(r _(s) ,r′,r _(d))F(r′)dr′  (7)

where W is a function which represents the expected contribution of eachpoint of the tissue volume V to the measurement at r_(d) for a sourcedistribution centered at r_(s) and is usually termed the weightfunction. Both r_(s) and r_(d) may be located either on the surface orvery close to it. The above equation may be discretized and solved as asystem of equations since there is a discreet number of sources anddetectors:

[F]_(j)={[W]_(j) ^(sd)}⁻¹[U _(nw)]_(sd)  (8)

Depending on the choice for the weight function, information on theinhomogeneities of the volume V may be included, in which case the aboveequation is non-linear, or the system of equations may be linearized,arriving at the Born Approximation.

An application of this example to simulated data, using a tandembioluminescent and fluorescent construct in the presence of non-specificfluorescent background, is illustrated in FIG. 5.

Bioluminescence Weighted Fluorescence Data and Weight Matrix

In the previous example, consideration of the emission spectra of thebioluminescent reporters and the fluorescent probes was not introduced.In the specific case when both emit at different ranges of the spectrum,the actual emission profiles of both would be different. Introducing theweight due to the bioluminescence carries with it an assumption thatboth fluorescent and bioluminescent spectra are equivalent. In order tomake this assumption more general, and actually independent of thebioluminescence spectra altogether, an accurate approach is to weightboth the fluorescence and the weight matrix in the following manner:

U _(nw)(r _(s) ,r _(d))=h(r _(d))∫_(V) W(r _(s) ,r′,r _(d))F(r′)dr′  (9)

In mathematical terms, this would give the same result as when theweight h is not used, with the significant difference that this weightmakes zero those contributions which are not solely due to the specificprobe. When inverting the system of equations:

[F]_(j)={[Wh]_(j) ^(sd)}⁻¹[U _(nw)]_(sd)  (10)

Those measurements which are not included in the weight function h aretherefore not being used. In some embodiments, this approach yieldsquantitative data, regardless of the choice of h, as long as it is basedon the bioluminescence measurement and this measurement is non-zero.

Fluorescence Weighted Bioluminscence Reconstruction

Because the basis of the recovery of a 3D distribution is based on theability to modulate the light emission by introducing a spatialdependence, this light emission modulation may be introduced into thebioluminescence measurement by making use of the fluorescencemeasurements. To that end, one approach would be to measure the averagenormalized fluorescence emitted, weighted by the bioluminescencemeasurement for each source, that is:

$\begin{matrix}{{I_{w}^{bio}\left( r_{i} \right)} = \frac{\Sigma_{j}^{Nd}\left\{ \left| \frac{U^{fl}\left( {r_{i},r_{j}} \right)}{U^{exc}\left( {r_{i},r_{j}} \right)} \middle| {U^{bio}\left( r_{j} \right)} \right. \right\}}{{mean}\mspace{14mu} \left\{ {U^{bio}\left( r_{j} \right)} \right\}}} & (11)\end{matrix}$

where N_(d) is the number of detectors. In doing the above, a fixedintensity distribution is determined, given by the bioluminescenceimage, and an intensity modulated by the excitation source given byI_(w) ^(bio)(r_(i)). As in the case of fluorescence, the 3D distributionof bioluminescence may now be recovered by:

U _(w) ^(bio)(r _(s) ,r _(d))=U ^(bio)(r _(d))I _(w) ^(bio)(r_(s))=∫_(V) W(r _(s) ,r′,r _(d))B(r′)dr′  (12)

where now B represents the spatial distribution of substrate plus enzymeconcentration of the bioluminescent reaction, and W is as defined forthe fluorescence tomography case. Following the previous example,inverting the system of equations:

[B]_(j)={[W]_(j) ^(sd)}⁻¹[U _(w) ^(bio)]_(sd)  (13)

Experiment Design and Acquisition of Data

In the previous three examples, an assumption is made that measurementsare acquired of the bioluminescence signal as well as of thefluorescence excitation light source and the fluorescence signal. Thisexample describes how these data may be acquired.

The first step is the preparation of the animal for imaging. Thebioluminescent reporter and fluorescent probe is introduced into theanimal, either by injection of a combined bioluminescent and fluorescentprobe, by injection of separate bioluminescent and fluorescent probes,and/or by injection of a fluorescent probe into an animal model thatalready contains bioluminescent cells, e.g. luciferase-expressingtransgenic cells. In any of these cases, a bioluminescence substrate,e.g. luciferin, is also injected at a time appropriate to enable bothfluorescence and bioluminescence imaging sequentially. Because thepharmacokinetics (PK) of the probes and substrate may be different,these injections may need to be performed at different times.

After acquiring a noise image, the bioluminescence image is acquired, asare images at the excitation and fluorescence emission wavelengths usingeach light source. As an alternative to the bioluminescence image, afluorescence image without an external light source could be acquired,with the fluorescent emission produced by means of radiative transferfrom the bioluminescent emitters to the fluorescent acceptors.Bioluminescent stimulation of fluorescence by radiative transfer hasbeen described using the acronym FUEL by Dragavon et al. (2010) Proc. ofSPIE Vol. 7902, in an in vitro context. This approach has the dualadvantages that the spectral mismatch described in the second exampleabove is eliminated and the measured fluorescence would be limited tothat stimulated directly by the bioluminescent reporter.

Finally, the fluorescence images would be normalized by theircorresponding excitation images as described, for example, in U.S. Pat.Nos. 6,615,063; 7,383,076.

In certain embodiments, the methods of the present invention are usefulwith optical imaging modalities and measurement techniques including,but not limited to: endoscopy; fluorescence endoscopy; luminescenceimaging; bioluminescence tomography, time resolved transmittanceimaging; transmittance imaging; nonlinear microscopy; confocal imaging;acousto-optical imaging; photoacoustic imaging; reflectancespectroscopy; spectroscopy; coherence interferometry; interferometry;optical coherence tomography; diffuse optical tomography andfluorescence mediated molecular tomography (continuous wave, time domainfrequency domain systems and early photon), and measurement of lightscattering, absorption, polarization, luminescence, fluorescencelifetime, quantum yield, and quenching.

Commercially available systems that can be used to employ the systemsand methods described herein include, but are not limited to, thefollowing: eXplore Optix™, Optix® and SoftScan® (ART—Advanced ResearchTechnologies, Canada), NightOWL® II LB (Berthold Technologies, Germany),NanoSPECT™, NanoPET/CT™ and HiSPECT® (Bioscan, Washington, D.C.), PhotonImager™, Beta Imager™, Micro Imager, Gamma Imager (Biospace Lab,France), Maestro® FLEX and Nuance® FLEX (Cambridge Research andInstrumentation—Cri®, Woburn, Mass.), LightSpeed™, BrightSpeed™ and MRSigna® Series, eXplore Series, Triumph™ (GE® Healthcare, UnitedKingdom), Kodak® In-Vivo Imaging FX Systems, Kodak® In-VivoMultispectral Imaging FX Systems and Kodak® Image Station 4000 series(KODAK® and Carestream®, Rochester, N.Y.), Aquacosmos® (Hamamatsu,Japan), CTLM® and LILA Imaging Systems (Imaging Diagnostic Systems—IMDS,Plantation, Fla.), Odyssey® Infrared Imaging System, Pearl® Imager(LI-COR, Lincoln, Nebr.), IMRIS® Neuro System (IMRIS®, Canada),Cellvizio® (Mauna Kea Technologies, France), SPY® and SPY®-TMR Systems,HELIOS™, LUNA™, PINPOINT®, and OPTTX® Imaging Systems (Novadaq, Canada),DYNOT Imaging System (NIRx, Glen Head, New York), OV100 and IV100(Olympus Corporation, Japan), Lumazone® (Photometrics, Tucson, Ariz.),and IVIS® Systems, IVIS® 3D, IVIS® Kinetics, IVIS® Spectrum and IVIS®Lumina (Xenogen®, Alamaeda, Calif. and Caliper® Life Sciences,Hopkinton, Mass.), iBox® (UVP, Upland, Ca), and VisEn FMT-1, VisEn FMT1500™, and VisEn FMT2500™ LX (VisEn™ Medical, Bedford, Mass.).

FIG. 6 shows a schematic of a diffuse optical tomography imaging system600 that may be used in various embodiments described herein. Theoptical data collection system 602 includes one or more light sources604, for example, a laser providing near infrared excitation light at agiven wavelength, e.g., in the 650 nm to 900 nm range. Multiplewavelengths of excitation light may be used. The light source(s) 604illuminate the object 612 within the imaging chamber 610, and light fromthe object is detected by one or more light detector(s) 606. The lightdetector(s) 606 may be, for example, a CCD camera, e.g., a time-gatedintensified CCD camera (iCCD).

The imaging chamber 610 houses the object 612 being imaged. The imagingchamber may be designed to allow direct optical contact between theobject 612 and the light source(s) 604 and detector(s) 606, or there maybe free space (e.g., air) between the object 612 and the light source(s)604 and/or detector(s) 606.

A data processor 608 can be part of the optical data collection system602 to pre-process or process image data, and/or a separate imageprocessor 614 can be used to process image data. Data corresponding tothe excitation light transmitted through the object 612, i.e., thetransilluminating excitation light, can be processed along with thefluorescent light and/or bioluminescent light images for improvedaccuracy. Background light can be corrected for and calibrationperformed for repeatability and accuracy of imaging results. A forwardproblem is established and tomographic reconstruction performed toprovide the tomographic image. Per methods described herein, thedetected bioluminescent light, detected fluorescent light, and/ordetected excitation light can be processed to provide a resultingtomographic image. The resulting tomographic image can be displayed on astandard display 616. Optical filters may be used.

Systems of the invention may include a computer which executes softwarethat controls the operation of one or more instruments, and/or thatprocesses data obtained by the system. The software may include one ormore modules recorded on machine-readable media such as magnetic disks,magnetic tape, CD-ROM, and semiconductor memory, for example. Themachine-readable medium may be resident within the computer or can beconnected to the computer by a communication link (e.g., access viainternet link). However, in alternative embodiments, one can substitutecomputer instructions in the form of hardwired logic for software, orone can substitute firmware (i.e., computer instructions recorded ondevices such as PROMs, EPROMS, EEPROMs, or the like) for software. Theterm machine-readable instructions as used herein is intended toencompass software, hardwired logic, firmware, object code and the like.

The computer is preferably a general purpose computer. The computer canbe, for example, an embedded computer, a personal computer such as alaptop or desktop computer, or another type of computer, that is capableof running the software, issuing suitable control commands, and/orrecording information in real-time. The computer may include a displayfor reporting information to an operator of the instrument (e.g.,displaying a tomographic image), a keyboard for enabling the operator toenter information and commands, and/or a printer for providing aprint-out, or permanent record, of measurements made by the system andfor printing diagnostic results, for example, for inclusion in the chartof a patient. In certain embodiments, some commands entered at thekeyboard enable a user to perform certain data processing tasks. Incertain embodiments, data acquisition and data processing are automatedand require little or no user input after initializing the system.

In certain embodiments, the invention features an in vivo imaging methodfor selectively imaging a subject containing two or more imaging probessimultaneously, wherein two or more imaging probes are administered to asubject, either at the same time or sequentially. The imaging probes canbe any combination of optical or other imaging agents. A single imagingagent may serve as both an optical and other imaging modality agent,e.g., dual imaging agent. The method therefore allows the recording ofmultiple biological processes, functions or targets. The methods of theinvention can be used to determine a number of indicia, includingtracking the localization of the imaging probes in the subject over timeor assessing changes or alterations in the metabolism and/or excretionof the imaging probes in the subject over time. The methods can also beused to follow therapy for such diseases by imaging molecular events andbiological pathways modulated by such therapy, including but not limitedto determining efficacy, optimal timing, optimal dosing levels(including for individual patients or test subjects), pharmacodynamicparameters, and synergistic effects of combinations of therapy.

In certain embodiments, this invention can be used with other imagingapproaches such as the use of devices including but not limited tovarious scopes (microscopes, endoscopes), catheters and optical imagingequipment, for example computer based hardware for tomographicpresentations.

Embodiments of the invention can be used, for example, to help aphysician, surgeon, or other medical personnel or researcher to identifyand characterize areas of disease, such as arthritis, cancers,metastases or vulnerable or unstable plaque, to distinguish diseased andnormal tissue, such as detecting tumor margins that are difficult todetect.

In certain embodiments, the methods of the invention can be used in thedetection, characterization and/or determination of the localization ofa disease, especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and monitoringand guiding various therapeutic interventions, such as surgicalprocedures, and monitoring and/or development of drug therapy anddelivery, including cell based therapies. The methods of the inventioncan also be used in prognosis of a disease or disease condition. Withrespect to each of the foregoing, examples of such disease or diseaseconditions that can be detected or monitored (before, during or aftertherapy) include inflammation (for example, inflammation caused byarthritis, for example, rheumatoid arthritis), cancer (for example,colorectal, ovarian, lung, breast, prostate, cervical, testicular, skin,brain, gastrointestinal, pancreatic, liver, kidney, bladder, stomach,leukemia, mouth, esophageal, bone, including metastases), cardiovasculardisease (for example, atherosclerosis and inflammatory conditions ofblood vessels, ischemia, stroke, thrombosis, disseminated intravascularcoagulation), dermatologic disease (for example, Kaposi's Sarcoma,psoriasis, allergic dermatitis), ophthalmic disease (for example,macular degeneration, diabetic retinopathy), infectious disease (forexample, bacterial, viral, fungal and parasitic infections, includingAcquired Immunodeficiency Syndrome, Malaria, Chagas Disease,Schistosomiasis), immunologic disease (for example, an autoimmunedisorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetesmellitus, lupus erythematosis, myasthenia gravis, Graves disease),central nervous system disease (for example, a neurodegenerativedisease, such as Parkinson's disease or Alzheimer's disease,Huntington's Disease, amyotrophic lateral sclerosis, prion disease),inherited diseases, metabolic diseases, environmental diseases (forexample, lead, mercury and radioactive poisoning, skin cancer),bone-related disease (for example, osteoporosis, primary and metastaticbone tumors, osteoarthritis), neurodegenerative disease, andsurgery-related complications (such as graft rejection, organ rejection,alterations in wound healing, fibrosis or other complications related tosurgical implants). The methods of the invention can therefore be used,for example, to determine the presence of tumor cells and localizationand metastases of tumor cells, the presence and localization ofinflammation, including the presence of activated macrophages, forinstance in atherosclerosis or arthritis, the presence and localizationof vascular disease including areas at risk for acute occlusion (e.g.,vulnerable plaques) in coronary and peripheral arteries, regions ofexpanding aneurysms, unstable plaque in carotid arteries, and ischemicareas, and stent thrombosis. The methods and compositions of theinvention can also be used in identification and evaluation of celldeath, injury, apoptosis, necrosis, hypoxia and angiogenesis. Themethods and compositions of the invention can also be used in formonitoring trafficking and localization of certain cell types, includingT-cells, tumor cells, immune cells, stem cells, and other cell types. Inparticular, this method may be used to monitor cell based therapies. Themethods and compositions of the invention can also be used as part ofphotodynamic therapy, including imaging, photoactivation and therapymonitoring.

In certain embodiments, the systems and methods described herein can beused to image endogenous fluorescence in a subject. For example, a geneencoding a fluorescent protein, such as green, red or infraredfluorescent protein, can be included adjacent to a gene of interest thatis to be expressed in an animal or human subject using standard genetherapy and transgenic techniques. The expression of the gene ofinterest can be determined indirectly by imaging the fluorescentprotein. If this protein is expressed, then the gene of interest hasalso been expressed. Fluorescence properties of endogenous fluorescentproteins are described in Giepmans et al., Science, 312: 217-224, 2006;Shaner et al., Nature Methods 2:905-909, 2005; and Zhang et al., Nat.Rev. Mol. Biol. 3: 906-918, 2002; Ai et al., Biochemistry 46:5904-5910,2007; Shaner et al., Nat. Biotech 22:1567-1572, 2004; Campbell et al.,Proc. Nat. Acad. Sci. 99:7877-7882, 2002; Heikal et al. Proc. Nat. Acad.Sci. 97:11996-12001, 2000; Baird et al., Proc. Nat. Acad. Sci.97:11984-11989, 2000; Tsien, Ann. Rev. Biochem. 67:509-44, 1998; Heim etal., Curr. Biol. 6:178-182, 1996; Cubitt et al., Trends Biochem Sci.11:448-455, 1995; Heim et al., Proc. Nat. Acad. Sci 91:12501-12504,1994; the relevant text incorporated by reference herein.

In certain embodiments, the systems and methods described herein can beused with systems and methods described in U.S. Pat. Nos. 6,615,063;7,383,076; and 8,190,241, which relate to extracting quantitative,three-dimensional molecular information from living mammals and patientsusing fluorochromes and optical tomographic imaging methods. Thesepatents describe the use of detected fluorescent images together withimages of transilluminating excitation light to obtain a more accuratetomographic image of a target region.

In certain embodiments, the systems and methods described herein can beused with systems and methods described in U.S. Patent ApplicationPublication No. US 2007/0238957, which relates to the use of X-ray datato account for different tissue types and densities in diffuse opticaltomography.

In certain embodiments, the systems and methods described herein can beused with systems and methods described in U.S. Patent ApplicationPublication No. 2005/0283071. U.S. Pat. No. 8,170,651, and U.S. PatentApplication Publication No. 2013/0114069, which relate to computationalsystems and methods for imaging volumes with arbitrary geometries innon-contact tomography.

In certain embodiments, the systems and methods described herein can beused with systems and methods described in U.S. Pat. No. 8,401,618,which relates to in vivo imaging systems and methods for tomographicimaging employing a hybrid approach for fast reconstruction. In thishybrid approach, one or more subsets of large tomographic datasets areselected in frequency space (e.g., Fourier space) while one or moresubsets are maintained in real space, then the weight matrix is invertedto obtain the tomographic representation of a region of interest withinthe subject in real space. This achieves fast computational times whilemaintaining good tomographic reconstruction performance.

In certain embodiments, the systems and methods described herein can beused with systems and methods described in U.S. Pat. No. 8,401,619,which relates to in vivo imaging systems and methods employing a virtualindex matching technique.

Imaging Probes (Fluorescent Species)

The imaging system and method can be used with a number of differentfluorescent imaging probes (or, as in embodiments using a tandembioluminescent reporter/fluorescent probe, the fluorescent speciesthereof), for example, (1) probes that become activated after targetcontact (e.g., binding or interaction) (Weissleder et al., NatureBiotech., 17:375-378, 1999; Bremer et al., Nature Med., 7:743-748, 2001;Campo et al., Photochem. Photobiol. 83:958-965, 2007); (2) wavelengthshifting beacons (Tyagi et al., Nat. Biotechnol., 18:1191-1196, 2000);(3) multicolor (e.g., fluorescent) probes (Tyagi et al., Nat.Biotechnol., 16:49-53, 1998); (4) probes that have high binding affinityto targets, e.g., that remain within a target region while non-specificprobes are cleared from the body (Achilefu et al., Invest. Radiol.,35:479-485, 2000; Becker et al., Nature Biotech. 19:327-331, 2001; Bujaiet al., J. Biomed. Opt. 6:122-133, 2001; Ballou et al. Biotechnol. Prog.13:649-658, 1997; and Neri et al., Nature Biotech. 15:1271-1275, 1997);(5) quantum dot or nanoparticle-based imaging probes, includingmultivalent imaging probes, and fluorescent quantum dots such as amineT2 MP EviTags® (Evident Technologies) or Qdot® Nanocrystals(Invitrogen™); (6) non-specific imaging probes e.g., indocyanine green,AngioSense® (VisEn Medical); (7) labeled cells (e.g., such as cellslabeled using exogenous fluorophores such as VivoTag™ 680,nanoparticles, or quantum dots, or by genetically manipulating cells toexpress fluorescent or luminescent proteins such as green or redfluorescent protein; and/or (8) X-ray, MR, ultrasound, PET or SPECTcontrast agents such as gadolinium, metal oxide nanoparticles, X-raycontrast agents including iodine based imaging agents, or radioisotopicform of metals such as copper, gallium, indium, technetium, yttrium, andlutetium including, without limitation, 99m-Tc, 111-In, 64-Cu, 67-Ga,186-Re, 188-Re, 153-Sm, 177-Lu, and 67-Cu. The relevant text of theabove-referenced documents are incorporated by reference herein. Anothergroup of suitable imaging probes are lanthanide metal-ligand probes.Fluorescent lanthanide metals include europium and terbium. Fluorescenceproperties of lanthanides are described in Lackowicz, 1999, Principlesof Fluorescence Spectroscopy, 2^(nd) Ed., Kluwar Academic, New York, therelevant text incorporated by reference herein. In the methods of thisinvention, the imaging probes can be administered systemically orlocally by injecting an imaging probe or by topical or other localadministration routes, such as “spraying”. Furthermore, imaging probesused in the application of this invention can be conjugated to moleculescapable of eliciting photodynamic therapy. These include, but are notlimited to, Photofrin, Lutrin, Antrin, aminolevulinic acid, hypericin,benzoporphyrin derivative, and select porphyrins.

In general, fluorescent quantum dots used in the practice of thisinvention are nanocrystals containing several atoms of a semiconductormaterial (including but not limited to those containing cadmium andselenium, sulfide, or tellurium; zinc sulfide, indium-antimony, leadselenide, gallium arsenide, and silica or ormosil), which have beencoated with zinc sulfide to improve the properties of the fluorescentagents.

In particular, molecular imaging probes are a preferred type of imagingprobe. A molecular imaging probe is a probe that is targeted to abiomarker, molecular structure or biomolecule, such as a cell-surfacereceptor or antigen, an enzyme within a cell, or a specific nucleicacid, e.g., DNA, to which the probe hybridizes. Biomolecules that can betargeted by imaging probes include, for example, antibodies, proteins,glycoproteins, cell receptors, neurotransmitters, integrins, growthfactors, cytokines, lymphokines, lectins, selectins, toxins,carbohydrates, internalizing receptors, enzyme, proteases, viruses,microorganisms, and bacteria.

In certain embodiments, optical imaging probes have excitation andemission wavelengths in the red and near infrared spectrum in the range550-1300 or 400-1300 nm or about 440 and about 1100 nm, between about550 and about 800 nm, between about 600 and about 900 nm. Use of thisportion of the electromagnetic spectrum maximizes tissue penetration andminimizes absorption by physiologically abundant absorbers such ashemoglobin (<650 nm) and water (>1200 nm). Optical imaging probes withexcitation and emission wavelengths in other spectrums, such as thevisible and ultraviolet light spectrum, can also be employed in themethods of the present invention. In particular, fluorophores such ascertain carbocyanine or polymethine fluorescent fluorochromes or dyescan be used to construct optical imaging agents, e.g. U.S. Pat. No.6,747,159 to Caputo et al. (2004); U.S. Pat. No. 6,448,008 to Caputo etal. (2002); U.S. Pat. No. 6,136,612 to Della Ciana et al. (2000); U.S.Pat. No. 4,981,977 to Southwick, et al. (1991); U.S. Pat. No. 5,268,486to Waggoner et al. (1993); U.S. Pat. No. 5,569,587 to Waggoner (1996);U.S. Pat. No. 5,569,766 to Waggoner et al. (1996); U.S. Pat. No.5,486,616 to Waggoner et al. (1996); U.S. Pat. No. 5,627,027 to Waggoner(1997); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S. Pat. No.5,877,310 to Reddington, et al. (1999); U.S. Pat. No. 6,002,003 to Shen,et al. (1999); U.S. Pat. No. 6,004,536 to Leung et al. (1999); U.S. Pat.No. 6,008,373 to Waggoner, et al. (1999); U.S. Pat. No. 6,043,025 toMinden, et al. (2000); U.S. Pat. No. 6,127,134 to Minden, et al. (2000);U.S. Pat. No. 6,130,094 to Waggoner, et al. (2000); U.S. Pat. No.6,133,445 to Waggoner, et al. (2000); U.S. Pat. No. 7,445,767 to Licha,et al. (2008); U.S. Pat. No. 6,534,041 to Licha et al. (2003); U.S. Pat.No. 7,547,721 to Miwa et al. (2009); U.S. Pat. No. 7,488,468 to Miwa etal. (2009); U.S. Pat. No. 7,473,415 to Kawakami et al. (2003); also WO96/17628, EP 0 796 111 B1, EP 1 181 940 B1, EP 0 988 060 B1, WO98/47538, WO 00/16810, EP 1 113 822 B1, WO 01/43781, EP 1 237 583 A1, WO03/074091, EP 1 480 683 B1, WO 06/072580, EP 1 833 513 A1, EP 1 679 082A1 WO 97/40104, WO 99/51702, WO 01/21624, and EP 1 065 250 A1; andTetrahedron Letters 41, 9185-88 (2000).

Exemplary fluorochromes for optical imaging probes include, for example,the following: Cy5.5, Cy5, Cy7.5 and Cy7 (GE® Healthcare);AlexaFluor660, AlexaFluor680, AlexaFluor790, and AlexaFluor750(Invitrogen); VivoTag™680, VivoTag™-5680, VivoTag™-S750 (VisEN Medical);Dy677, Dy682, Dy752 and Dy780 (Dyomics®); DyLight® 547, and/or DyLight®647 (Pierce); HiLyte Fluor™ 647, HiLyte Fluor™ 680, and HiLyte Fluor™750 (AnaSpec®); IRDye® 800CW, IRDye® 800R5, and IRDye® 700DX (Li-Cor®);ADS780WS, ADS830WS, and ADS832WS (American Dye Source); XenoLight CF™680, XenoLight CF™ 750, XenoLight CF™ 770, and XenoLight DiR (Caliper®Life Sciences); and Kodak® X-SIGHT® 650, Kodak® X-SIGHT 691, Kodak®X-SIGHT 751 (Carestream® Health).

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. The relevant teachings ofall the references, patents and patent applications cited herein areincorporated herein by reference in their entirety.

What is claimed is:
 1. A system for imaging a target region within adiffuse object, the system comprising: an excitation light source; anoptical imaging apparatus configured to direct light from the excitationlight source into the diffuse object at one or more of: (i) multiplelocations and (ii) multiple angles; one or more detectors, the one ormore detectors individually or collectively configured to at least oneof detect or measure: (i) one or more of bioluminescent orchemiluminescent light emitted from the object by a bioluminescentreporter in the target region of the object, (ii) fluorescent lightemitted by a fluorescent species in the target region of the diffuseobject as a result of excitation by the excitation light, and (iii)excitation light having been transmitted through the region of thediffuse object or having been reflected from the region of the diffuseobject; and a processor configured to process data corresponding to thedetected bioluminescent light, the detected fluorescent light, and thedetected excitation light to provide an image of the target regionwithin the diffuse object.
 2. The system of claim 1, wherein thebioluminescent reporter is endogenous.
 3. The system of claim 2, whereinthe bioluminescent reporter is expressed within the object by abioluminescent cell line.
 4. The system of claim 1, wherein thebioluminescent reporter is exogenously administered.
 5. The system ofclaim 1, further comprising a probe, the probe comprising at least oneof a fluorescent species or a jointly fluorescent/bioluminescentspecies, wherein the probe is administered to the object, and wherein atleast some of the bioluminescent reporter is substantially co-locatedwith at least some of the probe in the target region of the object. 6.The system of claim 5, wherein the bioluminescent reporter is alsopresent in the object at one or more locations that are notsubstantially co-located with the probe.
 7. The system of claim 1,further comprising a probe, the probe comprising at least one of afluorescent species or a jointly fluorescent/bioluminescent species,wherein the probe is administered to the object, and wherein the probecomprises both the fluorescent species and the bioluminescent reporterin tandem with the fluorescent species.
 8. The system of claim 1,wherein the diffuse object comprises living biological tissue.
 9. Thesystem of claim 1, wherein the diffuse object is located within aholder.
 10. The system of claim 9, wherein the image of the targetregion is a tomographic image, and wherein a surface of the object is atleast partially conformed by the holder such that the surface can becharacterized by a continuous function, thereby facilitating tomographicreconstruction.
 11. The system of claim 1, wherein the target region ofthe object is a three-dimensional region, and wherein the processor isconfigured to provide a tomographic image that corresponds to thefluorescent species in a three-dimensional target region.
 12. The systemof claim 11, wherein the tomographic image indicates concentration of aprobe as a function of position within the target region of the object.13. The system of claim 1, wherein the target region of the object is athree-dimensional region, and wherein the processor is configured toprovide a tomographic image that corresponds to the bioluminescentreporter in the three-dimensional target region.
 14. The system of claim1, wherein the processor is further configured to: weight datacorresponding to the detected fluorescent light with data correspondingto the detected bioluminescent light; and normalize data correspondingto the detected fluorescent light with data corresponding to thedetected excitation light.
 15. The system of claim 14, wherein theprocessor is further configured to: invert an associated weight matrixto obtain a tomographic image of the fluorescent species in the targetregion of the object.
 16. The system of claim 1, wherein the processoris further configured to: weight data corresponding to the detectedfluorescent light with data corresponding to the detected bioluminescentlight; weight an associated weight matrix with data corresponding to thedetected bioluminescent light; and invert the associated weight matrixto obtain a tomographic image of the fluorescent species in the targetregion of the object.
 17. The system of claim 1, wherein the processoris further configured to: weight data corresponding to the detectedbioluminescent light with data corresponding to the detected fluorescentlight normalized with data corresponding to the detected excitationlight; and invert an associated weight matrix to obtain a tomographicimage of the bioluminescent reporter in the target region of the object.18. The system of claim 1, wherein the bioluminescent light is detectedby the detector at a time different than when the fluorescent light isdetected by the detector.
 19. One or more non-transitorycomputer-readable media storing computer readable instructions that,when executed, cause a system to process data corresponding to detectedexcitation light, detected bioluminescent and/or chemiluminescent light,and detected fluorescent light to provide an image of a target regionwithin a diffuse object, by performing: determining an amount ofexcitation light corresponding to excitation light emitted, from anexcitation light source, into the diffuse object at multiple locationsand/or at multiple angles, wherein the detected excitation lightcorresponds to excitation light that has been either transmitted throughthe region of the diffuse object or reflected from the region of thediffuse object, determining an amount of bioluminescent and/orchemiluminescent light corresponding to bioluminescent and/orchemiluminescent light emitted, from the object, by a bioluminescentreporter in the target region of the object, determining an amount offluorescent light corresponding to fluorescent light emitted, by afluorescent species, in the target region of the diffuse object as aresult of excitation by the excitation light.
 20. An apparatus forreconstructing a tomographic representation of a probe within a targetregion of a diffuse object, the apparatus comprising: one or moreprocessors controlling operations of the apparatus; and a memory thatstores computer readable instructions that, when executed by the one ormore processors, configure the apparatus to: (a) establish a forwardmodel of excitation light propagation from an excitation light source tothe probe within the target region of the diffuse object and of emissionlight propagation from the probe to a detector using (i) datacorresponding to detected fluorescent light from the probe, (ii) datacorresponding to detected excitation light having been transmittedthrough the region of the diffuse object or having been reflected fromthe region of the diffuse object, and (iii) data corresponding todetected bioluminescent light emitted from a bioluminescent reporter,wherein at least some of the bioluminescent reporter is substantiallyco-located with at least some of the fluorescent light from the probe inthe target region of the object, and wherein the forward model isestablished as a weight matrix; and (b) invert the weight matrix toobtain the tomographic representation of the probe within the targetregion of the diffuse object.